The measurement of antioxidant capacity is one of the key analytical measures in the assessment of food ingredients with potential health benefits. Many methods are available to measure antioxidant potential or capacity such as the Oxygen Radical Absorbance Capacity (ORAC), the ferric ion reducing antioxidant power assay (FRAP), the Trolox Equivalence Antioxidant Capacity (TEAC) assay which is related to the ABTS⁺ method. Apak et al., (2008) and Pisoschi and Negulescu (2011) compare and review antioxidant methods and there is a brief summary of the methodology by Dai and Mumper (2010).

One of the earliest to be reported, the TEAC assay (Miller et al., 1993) relies on the long-lived radical anion ABTS^•+  being scavenged by the test antioxidants. Here, ABTS (2,2-azino-di-(3-ethylbenzothialozine-sulphonic acid)  is oxidized by peroxyl radicals or other oxidants to its radical cation, ABTS^·+ which is intensely coloured and relatively stable, but reacts rapidly with antioxidants, typically within 30 min. The antioxidant activity of a test material is usually referenced to the action of Trolox, by measuring the colour loss as the ABTS^·+ radical is mopped up.

A typical methodology for the ABTS method is:-

Usually, ABTS⁺ is produced by a reaction of equal amounts of  7 mM ABTS solution with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 10 to 15 hours. An extract of the solution to be assayed (0.5ml) is mixed with 3.5ml ethanol and 1 ml  ABTS⁺ solution. The solution is diluted further with ethanol at a ratio of 1:10 and the absorbance recorded at 734 nm versus a blank after 5 to 6 mins. A standard curve is prepared using different concentrations of Trolox and the results expressed as micromole TE per gram of fresh weight (fw) of product. A number of references are to be consulted for variants of the standard method but a modified form is often cited (Re et al., 1999).

The DPPH assay  is easier and more accurate than the TEAC version and relies on the reaction of antioxidants on 2,2-di(4-tertoctylphenyl)-1-picrylhydrazyl radical (DPPH) (Sanchez-Moreno, 2002). This assay relies on the loss of colour from DPPH at 515nm when reacting with various test materials (Bondet et al., 1997). The percentage of the remaining radical DPPH^· is proportional to the antioxidant concentration. The concentration causing a decrease in the initial DPPH level by 50% is defined as EC50. The only issue is when the test materials have absorption spectra overlapping that of DPPH.

The ORAC method measures antioxidant inhibition of peroxyl radical induced oxidation and this reflects a classical chain breaking reaction by antioxidants relying on H atom transfer (Ou et al., 2001). The assay relies on the peroxyl radical reacting with a fluorescent probe to form a non-fluorescent product. The antioxidant activity is determined by a decreased rate and amount of product formed over time. The ORAC assay has been adapted to measure lipophilic as well as hydrophilic antioxidants using a solution of 50% acetone/50% water (v/v) containing 7% randomly methylated α-cyclodextrin (RMCD) to solubilise the antioxidants (Huang et al., 2002).

The TRAP (total radical-trapping antioxidant parameter) assay involves the initiation of lipid peroxidation by generating water-soluble peroxyl radicals and is sensitive to all known chain breaking antioxidants.  It is widely used but relatively complex and time-consuming to perform, requiring a high degree of expertise and experience (Ghiselli et al., 2000). This assay measures the ability of antioxidant compounds to interfere with the reaction between peroxyl radicals generated by AAPH or ABAP [2,20-azobis(2-amidinopropane) dihydrochloride] and a target probe. The probe must be reactive with ROO radicals  at low concentrations as a specific requirement, and there must be a dramatic spectroscopic change between the native and oxidized probe (to maximize sensitivity), and no radical chain reaction beyond probe oxidation should occur. Typically, oxidation of the probe is followed spectrophotometrically (Bartosz et al., 1998) or by fluorescence (Ghiselli et al.,1995). TRAP values are expressed as a lag time or reaction time of the sample compared to the corresponding times for Trolox.

High antioxidant levels were implicated in the protective effects of various ingredients against the formation of diseases such as cancer and atherosclerosis. However, because of scepticism about the underlying mechanisms, especially the free radical theory, in the USA, the USDA removed its ORAC Database for Selected Foods from the NDL web-site. In their view, there was actually little evidence that antioxidant capacity had any relevance to specific ingredients such as polyphenols on specifically improving human health (USDA, 2012). In fact, there is some contradictory evidence based on the longevity of a commonly used research animal the nematode Caenorhabditis elegans which suggests free radical formation signals repair functions in organisms.

The author is actively involved with businesses wishing to understand more about antioxidants and how they are measured. Please contact me for further information.

References

Apak, R., Güçlü, K., Ozyürek, M., Çelik SE. 2008. Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay. Microchim Acta 160  pp. 413–9.

Bartosz, G., Janaszewska, A., Ertel, D., & Bartosz, M. (1998). Simple determination of peroxyl radical-trapping capacity. Biochem. Molecular Biology Int. 46, pp. 519–528.

Bondet, V., Brand-Williams, W., & Berset, C. (1997). Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Lebensmittel-Wissenschaft Technol., 30, pp. 609–615.

Dai, J., Mumper, R.J. (2010) Plant Phenolics: Extraction, Analysis and Their Antioxidant and Anticancer Properties. Molecules 15 pp. 7313-7352 doi:10.3390/molecules15107313

Ghiselli, A., Serafini, M.,Maiani, G., Azzini, E., & Ferro-Luzzi, A. (1995). A fluorescence-based method for measuring total plasma antioxidant capability. Free Radical Biology and Medicine, 18, pp. 29–36.

Ghiselli, A., Serafini, M., Natella, F., & Scaccini, C. (2000). Total antioxidant capacity as a tool to assess redox status: critical review and experimental data. Free Radical Biology and Medicine, 29, pp. 1106–1114.

Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J., & Deemer, E. (2002). Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated α-cyclodextrin as the solubility enhancer. J. Agric. Food Chem., 50, pp. 1815–1821.

Miller, N. J., Diplock, A. T., Rice-Evans, C., Davies, M. J., Gopinathan, V., & Milner, A. (1993). A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical Science, 84, pp. 407–412

Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem., 49, pp. 4619–4926

Pisoschi, A.M., Negulescu, G.P. (2011) Methods for Total Antioxidant Activity Determination: A Review. Biochem. Anal. Biochem. 1(1) pp. 1-10 http://dx.doi.org/10.4172/2161-1009.1000106

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic, Biol, Med.  26 pp. 1231–7.

Sanchez-Moreno, C. (2002). Review: Methods used to evaluate the free radical scavenging activity in foods and biological systems. FoodSci.Technol. Int., 8(3), pp. 121–137.

USDA (2012) Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods, Release 2 (2010) http://www.ars.usda.gov/services/docs.htm?docid=15866